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| Dissertation / PhD Thesis/Book | PreJuSER-53677 |
2006
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
Please use a persistent id in citations: http://hdl.handle.net/2128/2468
Report No.: Juel-4231
Abstract: In this work the influence of the PTS-sugars glucose, fructose and sucrose on growth as well as on lysine- and glutamate production in C. glutamicum was investigated. Growth rates, biomass yields and ethambutol induced glutamate production showed no differences on glucose, fructose or sucrose. In contrast, the lysine yield of C. glutamicum DM1730 on glucose was more than twofold increased as compared to the other tested sugars. Overexpression of the malic enzyme gene to improve the NADPH supply had no effect on the lysine yield whereas overexpresion of the fructose-1,6-bisphosphatase (fbp) gene resulted in a doubling of the lysine yield on sucrose. This result could be explained by the observation of a decreased intracellular concentration of fructose-1,6-bisphosphate, a competive inhibitor of the key enzymes of the pentose phopshate pathway, apon overexpression of fbp. That probably led to a higher carbon flux through the pentose phosphate pathway and thus to a better NADPH supply. A direct increase of the NADPH supply by overexpression of the transhydrogenase pntAB from E. coli led to increased lysine yields on all tested carbon sources. In order to identify the glucose, fructose and sucrose-stimulons, the global gene expression patterns during growth on these sugars were investigated using DNA-chips. The data revealed 53 genes repressed on LB-medium in the presence of any PTS-sugar. Some of these genes encoded transporters and enzymes for utilization of alternative carbon sources. Moreover, in the presence of fructose and sucrose the expression of genes necessary for adaptation to nitrogen starvation was increased. That effect could be explained by a concomitant repression of genes for utilization of oligopeptides, an important nitrogen source during growth on LB-medium. Thus, it was concluded that the nitrogen availability was limited in the presence of fructose and sucrose. To investigate regulative effects of PTS-sugars on utilization of alternitive carbon sources, carbon consumption rates were determined during growth of C. glutamicum on subtrate mixtures, consisting of a PTS-sugar and an organic acid. It was shown that the glucose consumption rate was inhibited fourfold by acetate, pyruvate and L-lactate while fructose und sucrose consumption was nat affected by these organic acids. During growth on fructose and L-lactate a diauxic growth pattern under preference of fructose was observed. To elucidate the mechanism of the fructose/L-lactate diauxie, the genetic regulation of the essential genes for L-lactate utilization was scrutinized. The so far unknown regulator NCgl2814 could be identified as a repressor of the L-lactate utilization operon NCgl2816-lldD. It could be shown that NCgl2814 binds -89 to -35 bp upstream of the translational start site of the NCgl2816-lldD operon. Binding of NCgl2814 was inhibited in the presence of 10 mM L-lactate. Based on this results a model for the regulation of the L-lactate metabolism was established. In this model in the absence of L-lactate NCgl2814 binds to the NCgl2816-lldD-promoter und represses its expression, while NCgl2814 dissociates from the promoter in the presence of L-lactate whichs results in an increased expression of the L-lactate utilization operon.
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